SELECTED SUMMARIES
October 1993
the light tioned,
of the muscle demonstration
cells with strong
strip
and pathophysiological
that
VZV
a coordinating
support
is confined
and inhibitory
studies to those
function
men-
would
provide
for our hypothesis.
The localization of achalasia to the esophagus suaests a local lesion rather than one in the central nervous system or the vagus nerve.
In our view, ganglion
the consequence
cell depletion
of esophageal
to sorne degree
in vigorous
is unlikely
distention, achalasia
because
before
to be simply
it can be shown
dilatation
has devel-
oped. We join Dr. Schulze-Delrieu cal role of VZV in achalasia confirm
or refute
myenteric ganglion
plexus
in hoping
and to localize which,
in specific
is a more
PROF.
to
in the esophageal
its presence
of course,
studies types
daunting
MICHAEL
of
ATKINSON
of Helsinki,
sense construct had a reduced growth rate. ODC transfected contrast
to the antisense
ODC
transfectants
Expression of human ODC by the pHPApr1 neo vector or the PMAMneo
vector
(dexamethasone
inducible)
cells with a lower efficiency
led to
of transfection
than the pLTRpoly/ODC
construct, reflecting different po-
tencies of the promoters.
When these vectors had the ODC
cDNA in the reverse direction,
no morphological
Additionally,
Southern
transfor-
blot analysis
indicated that human ODC was stably integrated into genoCells with the pLTRpoly/ODC
Helsinki,
Finland).
of Or-
PMAMneo
construct
ity, respectively.
ODC
activity
remained
versely, in the normal cells, ODC
26).
In this report from Auvinen et al., the authors studied the role of ornithine
decarboxylase
and oncogenesis.
A key regulator of polyamine
(ODC)
in transformation synthesis,
ODC is an enzyme whose expression is transiently increased and is constitutively However,
acti-
it is unclear
and
constructs had about 60% and 30% of that activ-
tion. Nature 1992;360:355-358
(November
had very high
ODC activity, whereas the pHPAprlneo
stable
vated during cell transformation.
and the un-
transfected cells.
nithine decarboxylase activity is critical for cell transforma-
upon growth factor stimulation
more rapidly than
and the cells with ODC anti-
cells could grow in low serum (0.5% fetal calf serum) in
constitutive
Auvinen M, Paasinen A, Andersson LC, et al. (Department University
controls,
mic DNA derived from the transfectants.
ORNITHINE DECARBOXYLASE: AN ONCOGENE? Pathology,
the untransfected
mation was evident.
task.
of antisense ODC. Cells
by ODC cDNA proliferated
transformed
etiologi-
by further
of VZV DNA
the presence
cells there,
that the possible
will be elucidated
were present with the transfection transformed
ganglion
1255
transfectants,
even
after
serum
high in all the starvation.
Con-
activity was negligible
after serum starvation. The authors then investigated whether the augmentation in ODC production
and its transforming
activity was asso-
ciated with changes
in tyrosine
upon the observation
that alpha-difluoromethyl
(DFMO),
phosphorylation,
based
ornithine
an inhibitor of ODC, causes a decrease in protein
whether ODC is a direct mediator or an innocent bystander
tyrosine phosphorylation.
Western blot analysis revealed a
of cell transformation.
protein of 130 kilodaltons,
which undergoes increased tyro-
tion,
the authors
To address that fundamental
inserted
human
ODC
ques-
complementary
sine phosphorylation
in the ODC transfectants
compared
DNA (cDNA) into three expression vectors with promoters
with the parental cells. The identity of this protein is un-
of different
clear. The authors further observed that blocking ODC by
cells.
strength
Concurrently,
marker (pSV2neo) resistant
and transfected control
plasmids
were transfected.
cells were isolated
growth and transforming
them into NIH3T3 and
a selectable
Thereafter,
neomycin-
after two weeks,
and their
properties were investigated.
The three expression vectors in which the human ODC cDNA was inserted were pLTRpoly
(contains long terminal
repeat [LTR] of Moloney murine leukemia virus as the promoter and the SV40 splicing and polyadenylation
signals),
pMAM neo (Rous sarcoma virus LTR enhancer
linked to
the dexamethasone-inducible
mouse mammary tumor virus
LTR promoter and SV40 splicing and polyadenylation plus the neomycin-resistance tains the human quence-l
P-actin
with enhancer
gene), and pHPAprlneo promoter
sites (con-
plus intervening
se-
like activity and the SV40 poly-
adenylation signal). All of these expression vectors were also assembled with human ODC cDNA in the antisense direction to serve as controls during transfections. When pLTRpoly/ODC was transfected into NIH3T3 cells, there was complete morphological transformation. In particular, the cells had no contact inhibition, ple foci
in monolayer
their actin filaments, colonies
formed multi-
cultures, displayed disintegration
of
and had acquired the ability to form
in soft agar. Conversely,
none of these features
DFMO inhibits transformation of rat fibroblasts by v-m. The authors conclude that ODC is a requisite for transformation,
and the degree of transformation
is tied to the
expression of ODC as indicated by the use of different expression vectors. Anything
but an innocent
bystander, the
authors suggest that ODC plays a significant
role in onco-
genesis.
Comment. mine
The
are normal
polyamines
in cell proliferation cells
depleted
of polyamines with
1988;48:759-774).
Based
lieved
that polyamines
charge
of DNA
amines
stabilize
RNA.
It is likely
which
polyamines
The
pathway
that
resume
on their
chemical
DNA
by phosphate RNA,
these
role
experiments:
polyamine-depleted growth
(Cancer
structures,
by neutralizing groups.
messenger
the negative
RNA,
and
provided
and protein
RNA,
synthesis to putrescine,
step in polyamine
from putrescine
in cells
decarboxylase.
by
synthesis.
is in large
part
catalyzes
is the first and
synthesis.
is catalyzed
polytransfer
the means
ODC
which
Res
it is be-
Additionally,
interactions
ornithine
of ornithine
sper-
DNA,
for polyamine
the rate-limiting
tion of spermidine
growing;
stimulate
by the enzyme
the conversion usually
stop
ribosomal
and
play a significant
by the following
polyamines stabilize
provided
spermidine,
of cells. They
as evidenced
cells supplemented
regulated
putrescine,
components
The forma-
by spermidine
syn-
1256
SELECTED
SUMMARIES
thase,
and in turn,
spermidine
thase.
The
derived
aminopropyl
from
of polyamine
specific
inhibitors,
(DFMO),
a highly
specific
The
mammalian
oncogene
the enzyme
often
amplification
have
been
of about
rat, mouse)
of ODC
and a predicted
(Trends
stimulation. increase
in ODC
synthesis
Another
ODC
malignancies Investigations
colon
carcinomas
1275-1281),
(Ann
Med
1984;311:80-83).
vated
in normal
administration
Surg
stimuli,
enhanced
fected
syn-
protein
1986;204:89-93;
of gastrin
(Am J Physiol
a marker
Thus,
of cellular
proteins
transformation
(Cancer
A key question
regulators
are indirect
cDNA
into
moters
of various
a mammalian
promoters
of
strengths.
can be transfected
cells such that the cDNA and protein
tyrosine
kinase
yet to be identiin the colon. is whether
transformation
is addressed
strategy
is the region elements
or introduced
into cultured
is transcribed
into RNA
parental,
or untransfected,
levels
in the transfected
in
in markedly
with the parental
ODC
cDNA)
activity
some
in the stably
cells ODC
a property
cells or
but with
activity
trans-
remained
of transformed
cells in
mechanisms Because
DFMO,
Western
tyrosine
antibodies
of ODC
protein
blot analysis
stably transfected
was performed
in protein
had undergone
cell transformation
phosphorylation
lysates
cells. This revealed
the cells transfected is under
induced
tyrosine
using
derived a unique
augmented
by
antiphospho-
from
parental
and
band of 130 kilodal-
tyrosine
with ODC cDNA.
were
is reduced
phosphorylation
The identity
in
of this protein
investigation.
The authors
conclude
that ODC is necessary
tion and that the degree
of transformation
level of ODC,
implying
perhaps
prerequisites
for
might
the injection
involve
into colon
cancer
ated,
the introduction
and
immortalized
a certain
transformation.
upon
the
level and activity
are
Potential
future
of tumors, of ODC
an agent
such
cells into nude
the introduction if transformation into
colonic
as SV40
epithelial
serve if these cells can be transformed.
This work may function
into how ODC
cell proliferation
and oncogenesis,
cellular
pathways
regulating
pro-
activity
represent
areas of exciting
ODC
of ODC is acceler-
large T antigen
providing
new insights
experiments
of the stably transfected
cell lines to measure
with
for cell transforma-
is dependent
especially
cells to ob-
is important
in GI cancers.
and polyamine
in
in abnormal The
expression
and
investigation.
that increase with
a
mammalian
and then transhigh
in cancers
cells compared
cells: rate of growth,
of the promoters
of DNA
vectors
observed
effects
in in
this resulted
compared
in these
growth was variability
There
transcription.
Expression
to mimic
certain
vectors growth,
because
starvation,
in the
expression
cells.
and ODC
three
cDNA
led to accelerated
of the strength
a given
contains
to initiate
one can achieve
measure
in the present
that
In this fashion,
or and tu-
is to insert
vector
enhancer
promoters.
they
the ODC
fibroblast with
anchorage-independent
(when
mice to assay the formation
in malignant
of transformation
polymerase
may also contain
may repin the
activity
malignant
question
after
especially
polyamines
The promoter
RNA
be ele-
efficiently,
of
vector.
culture.
tons that ]
1986;255:G597-G601;
ODC
expression
the use of some eukaryotic
lated into protein.
and
et al. A general
in binding
may mucosa
of certain,
biomarkers
This fundamental
by Auvinen
activity
Res 1989;49:5884-5888) ODC
1987;60:
colonic
Furthermore,
may regulate
about
direct they
of cells.
in
(N Engl
ODC level and activity
phosphorylation
fied, membrane
condi-
is enhanced
polyps
hyperproliferation,
transformation and tyrosine
of
cells, a mouse
All three
the antisense
Furthermore,
to take up and the expression
in the expression
NIH3T3
vectors
mRNA
high even after serum Possible
post-
in a variety
Cancer
adenomatous ODC
cells.
ability
Furthermore,
containing
as a function
in ODC
by the cell type that
vary in their
DNA.
and
cells with
variability
to a given
transfected
ODC activity
prolifer-
is resistant
that
is determined
directions.
expression
the parental
of ODC
premalignant
or normal
these properties
the selection,
of after
marker
in the sense direction
phenotype,
The efficiency
based upon
vectors
in the stably
explored.
that ODC activity
mucosa
soft agar
cDNA.
are performed
can be transfected
and the anti-sense
any
perturbation
the cDNA
stably transfected
with ODC cDNA
in the
of the cDNA,
without
(involves
by the promoter
generally
expression
the different
by the level
of the ODC
Additionally,
gastric
Life Sci 1990;46:777-784).
morigenesis.
the
to be due to an
of regulation
and their corresponding
The authors
the trans-
overexpressing
cell types
is influenced
bound-
and serum
loop as determined
but also in benign
constitute
gastrin
is believed
of
before
mitogenic
has been observed
have shown
the cDNA
No. 4
direction
of techniques.
media)
added
alone
measurements
of a selectable
exogenously
sense
is at the level of ODC protein
expression
express
the ODC
phases
transition
of clones
involve
into a cell involves
by a variety
drug in the tissue culture
transformed
factor,
modification
cotransfection
controls
or opposite,
vector
DNA
the desired
Vol. 105,
is no transcription
or stable transfection
cell line that
1 amino acids
at the G2/M
means
hours)
and analysis
different
with certain
certain
(usually
ation,
Useful
of the
of exogenous
48-72
transcript
encoding to 46
phase
synthesis
feedback
involves
tions.
malignant
gene
(Genomics
(Mol Cell Biol 1988;9:5484-5490).
Deregulated human
frame
growth
This regulation
translationally
Of
of the ODC
corresponds
following
ODC
mRNA.
thesis or perhaps
ODC.
one ODC mRNA
at the Gl/S
increases
is by a negative
of polyamines.
is near
to hydroxyurea
and it also increases
ester, epidermal
Increased
transfection
is used as different
the gene for
mass of 51 kilodaltons.
peaks
synthesis
such as phorbol
2
retinoblas-
Furthermore, M2 subunit
which
11
The chromosomal
of ODC correlates
synthesis,
contains
two ODC gene
in that it is near N-m_yc,
The open reading
activity
the cell cycle. ODC ary. ODC
gene
contains
in neuroblastomas,
tissues express
molecular
The biological onset of DNA
ODC
genome
amplified
is 1383 nucleotides,
also
transient
reductase
Human
and
assays.
in the anti-sense,
such that there
of the cell membrane
in cells resistant
2.2 kilobases.
protein
RNA
by the use of
vector
inhibitor
of this gene as well as that
observed
1987;1:77-86).
cDNA
expression
irreversible
lung carcinomas.
ribonucletide
note,
DNA
of the cDNA
Transfection
of ODC is interesting
and small-cell
involved
tumorigenicity
fection
(dcSAM).
assisted
2 and 7, respectively.
(2p23ter)
a nuclear tomas,
study
been
notype,
are
2-difluoromethylornithine
The human
loci on chromosomes localization
whether
have
syn-
particular
(human,
and 12 exons.
activity
by spermine and spermine
S-adenosylmethionine
synthesis in
spermine
in spermidine
Sci 1990;15:153-158).
introns
resent
yields
groups
decarboxylated
Studies
Biochem
GASTROENTEROLOGY
alteration
levels of and then with
the
in phe-
Reply.
We would
like to thank
view of our findings future
experiments.
the ODC
and kind Noteworthy,
overexpressing
Dr. Rustgi
comments
our recent
studies
cells are also tumorigenic
tion of the pLTRpoly/ODC
transfected
NIH3T3
ously into nude mice gave rise to progressively all the cases, whereas
for his excellent
and outlining
the control
NIH3T3
re-
potential
indicate in vivo.
that Injec-
cells subcutane-
growing
tumors
cells were not tumori-
in